Annals of Botany 119 (6): 989-1000 (2017) 1 Original article 2 Title: 3 POLLEN TUBE ACCESS TO THE OVULE IS MEDIATED BY GLYCOPROTEIN 4 SECRETION ON THE OBTURATOR OF APPLE (MALUS X DOMESTICA, BORKH) 5 Authors: 6 Juan M. Losada1,2,3,4* 7 María Herrero4 8 1 Arnold Arboretum of Harvard University. 1300 Centre St. Boston, MA 02131. 9 2 Department of Organismic and Evolutionary Biology, Harvard University. 16 Divinity Ave, 10 Cambridge, MA 02138. 11 3 Department of Ecology and Evolutionary Biology, Brown University. 80 Waterman Street 12 Providence, RI 02912. 13 4Pomology Department, Aula Dei Experimental Station-CSIC. Avda Montañana 1005. 50059. 14 Zaragoza, Spain. 15 Running title: Secretions of the apple obturator 16 *E-mail address: [email protected] 17 1 Annals of Botany 119 (6): 989-1000 (2017) 1 ABSTRACT 2 Background and Aims Within the ovary, the obturator bridges the pathway of the pollen tube 3 from the style to the ovule. Despite its widespread presence among flowering plants, its function 4 has been only studied in a handful of species and the molecules involved in pollen tube-obturator 5 crosstalk have not been explored hitherto. This work evaluates the involvement of glucans and 6 glycoproteins on pollen tube growth in the obturator of apple flowers (Malus x domestica). 7 Methods Pollen tube kinetics was sequentially examined in the pistil and related to changes 8 occurring on the obturator using histochemistry and inmunocytochemistry. To discriminate 9 between changes in the obturator induced by pollen tubes from those developmentally regulated, 10 both pollinated and unpollinated pistils were examined. 11 Key Results Pollen tube growth rates were slow in the stigma, faster in the style and slow again 12 in the ovary. The arrival of pollen tubes to the obturator was concomitant with the secretion of 13 proteins, saccharides and glycoprotein epitopes belonging to extensins and arabinogalactan 14 proteins (AGPs). While some of these secretions - extensins and AGPs labeled by JIM13 - were 15 developmentally regulated, others - AGPs labeled by JIM8 - were elicited by the presence of 16 pollen tubes. Following pollen tube passage all these glycoproteins were depleted. 17 Conclusions Results show a timely secretion of glycoproteins on the obturator surface 18 concomitant with pollen tube arrival to this structure. The fact that secretion depletes following 19 pollen tube passage strongly suggests their role in regulating pollen tube access to the ovule. 20 Remarkably, both the regulation of the different glycoproteins secretion, as well as their 21 association with the performance of pollen tubes exhibit similarities with those observed in the 22 stigma, in line with their common developmental origin. 2 Annals of Botany 119 (6): 989-1000 (2017) 1 Key words: AGPs, arabinogalactan proteins, apple, extensins, glycoproteins, Malus x 2 domestica, obturator, ovary, pistil, pollen, syphonogamy, transmitting tract. 3 3 Annals of Botany 119 (6): 989-1000 (2017) 1 INTRODUCTION 2 From pollination to fertilization, regardless of the angiosperm taxon, pollen tubes travel 3 along three territories: the stigma, the style and the ovary (Heslop-Harrison, 1987, 2000; 4 Dresselhaus and Franklin-Tong, 2013). This apparent complicated scenario offers ample 5 opportunities for pistil support, but also control, of pollen tubes through regulated pistil 6 secretions along the pollen tube pathway (Herrero and Hormaza, 1996; Herrero, 2003). 7 Secretions at the stigma offer adequate support for pollen germination (Herrero and Dickinson, 8 1979; 1981; Hiscock and Allen, 2008, Losada and Herrero, 2012), but this support is not 9 indiscriminate (Hedhly et al, 2005). Along the style, pollen tube elongation relies on provisions 10 stored in the style (Herrero and Dickinson, 1979; Stephenson et al., 2003; Losada and Herrero, 11 2014). But this nutritional dependence also exerts a selective pressure on pollen tubes (Mulcahy, 12 1979; Hormaza and Herrero, 1992, 1994), with clear ecological and evolutionary implications 13 (Lankinen and Green, 2015; Hersh et al., 2015; Harder et al., 2016). When pollen tubes arrive in 14 the ovary they face a territory less well studied. While the molecular control of pollen tubes in the 15 ovary has been extensively studied at the ovules (Higashiyama and Takeuchi, 2015), the ovarian 16 pollen tube pathway between the stylar transmitting tissue and the ovule has remained neglected, 17 except in a handful of species (Arbeloa and Herrero, 1987; Herrero and Arbeloa, 1989; 18 Sornsathapornkul and Owens, 1999; Herrero, 2001, 2003; Nores et al., 2015). Interestingly 19 enough, the few available reports emphasize the obturator as a critical player in pollen tube 20 performance prior to syphonogamy. 21 Aside from the early description of the obturator by Dalmer in 1880, it has been almost 22 entirely overlooked in angiosperms. This is quite surprising, since the obturator has a consistent 23 presence across members of the major clades of flowering plants, including the early diverging 24 genus Kadsura (Schisandraceae; Lyew et al., 2007), mesangiosperm genera such as Persea 4 Annals of Botany 119 (6): 989-1000 (2017) 1 (Lauraceae; Sedgley and Anells, 1981), Artabotrys, Magnolia, Liriodendron (Magnoliaceae; 2 Matsui et al., 1993; Igersheim and Endress, 1997), Zygogynum (Winteraceae; Igersheim and 3 Endress, 1997), as well as at least seventy families of monocots and dicots (Stevens, 2001 and 4 refs.; Endress and Mathews, 2006; Nores et al., 2015). Unfortunately, the functionality of the 5 obturator is difficult to evaluate, since it is buried in the ovary and requires a sequential 6 examination of a developmental process. But as a matter of fact, the obturator is a critical 7 territory for pollen tubes as it bridges the stylar transmitting tissue with the ovule entrance (Juel, 8 1919; Tilton and Horner, 1980; Tilton et al., 1984; Endress, 1994). This relationship is 9 straightforward in species with orthotropous ovules in which the ovule entrance faces towards the 10 style (Arbeloa and Herrero, 1987; Herrero and Arbeloa, 1989). In contrast, a sharper switch in 11 directionality occurs in species with anatropous ovules, in which the ovule entrance is opposite 12 that of the style, and pollen tubes have to turn 90º to 180º towards the micropyle (Endress, 2015; 13 Nores et al., 2015). While this directional switch occurs on the obturator surface, its role on 14 pollen tube directionality towards the ovule has scarcely been studied. In peach flowers, pollen 15 tube growth is arrested upon arrival at the obturator until this structure enters a secretory phase, 16 then the pollen tube further resumes growth (Arbeloa and Herrero, 1987). In other species, the 17 obturator enters the secretory phase before pollen tube arrival (González et al. 1996). Still, in all 18 instances examined the provision of a secretion in the obturator appears to be a prerequisite for 19 pollen tube passage, but the timing and nature of this secretion remains enigmatic for most 20 species. 21 Glycoproteins are a major player in the communication between developing pollen tubes 22 and pistil tissues in angiosperms, especially as a form of pollen tube recognition within the style 23 (McClure et al., 2011; Pereira et al., 2015). In Nicotiana, pollen tube nourishment has been 24 further demonstrated by a deglycosylation gradient of glycoproteins along the transmitting tract 5 Annals of Botany 119 (6): 989-1000 (2017) 1 as pollen tubes elongate through, consistent with the idea of a basipetal maturation of the pistil 2 (Wu et al., 1995; 2000). Among glycoproteins, arabinogalactan proteins (AGPs) ubiquitously 3 localize in the pollen tube pathway of angiosperms (Cheung et al., 1995; Sánchez et al, 2004; 4 Sage et al., 2009; Pereira et al., 2014). Indeed, they play key roles during the initial stages of the 5 progamic phase (Losada and Herrero, 2012; Suárez et al., 2013; Losada et al., 2014). Curiously, 6 species with large styles such as apple exhibit a variable glycoprotein topology along the pistil. In 7 the stigma of the apple flower, AGPs set the time for pollen receptivity (Losada and Herrero, 8 2012). Conversely, in the contiguous transmitting tract of the style, glycoproteins extensins and 9 preformed beta glucans preclude a rapid pollen tube elongation (Losada and Herrero, 2014). This 10 is where glycoprotein topology analysis stops, leaving the adjacent tissues of the ovary largely 11 unexplored. The concurrent participation of AGPs along other stretches of the pollen tube 12 pathway suggests that they could well be a component of the obturator secretion. Therefore, this 13 work evaluates pollen tube growth in relation to changes in the obturator of the apple (Malus x 14 domestica) flower, with emphasis on the immunolocalization of glycoproteins. 15 16 MATERIALS AND METHODS 17 Plant material 18 Flowers from apple trees cv Golden Delicious Spur grown in the province of Huesca 19 (Spain), 461m above sea level were collected over two years. One day before anthesis (flower 20 opening), flowers were emasculated and left for 24h until hand pollination. Since apples are self- 21 incompatible, anthers from the compatible cv Royal Gala were collected from flowers at the same 22 developmental stage and left to dry on paper at room temperature (20-25ºC) for 24-48h until 23 dehiscence. Pollen was sieved through a metallic mesh with a diameter pore of 0.26 mm, and 24 used to pollinate emasculated flowers, whereas leaving a second batch of flowers unpollinated. 6 Annals of Botany 119 (6): 989-1000 (2017) 1 2 Observation of pollen tube growth 3 In order to monitor the arrival of pollen tubes to the different tissues along the pistil, 4 gynoecia were fixed daily from anthesis to seven days after pollination (DAP). Five gynoecia 5 from each batch were fixed in FAA (formalin: acetic acid: 70% ethanol) (1:1:18) (Johansen, 6 1940) for at least five hours.